文摘
Glutamine 105 in the third transmembrane helix of thethyrotropin-releasing hormone receptor(TRH-R) occupies a position equivalent to a conserved negativelycharged residue in receptors for biogenicamines where it acts as counterion interacting with the cationic aminemoiety of the ligand. Maximumlevels of response to TRH in oocytes expressing wild-type TRH-Rs wereindistinguishable from those ofoocytes expressing receptors mutated to Glu, Asn, or Asp in position105. However, the EC50 values foractivation of oocyte responses increased more than 500 times in oocytesexpressing mutant Glu105 receptors,in which the amido group of Gln105 has been removed bysite-directed mutagenesis. Charge effects donot seem to be involved in the huge effect of mutatingGln105 to Glu, since mutation of Gln105 toAspinduces only a 15-fold increase in EC50. Furthermore,no change in EC50 is observed after mutationofAsn110 to Asp. The affinity shift (identified bychanges in EC50 values for systems of comparableefficacy)in Glu105 mutant receptors was partially recovered inoocytes expressing Asn105 mutant receptors.Theseresults and those obtained after substitution of Lys, Leu, Tyr, and Serfor Gln105 suggest that the presenceand the correct position of the Gln hydrogen bond-donor amido groupare important for normalfunctionality of the receptor. In wild type or Asp105mutant receptors showing the same maximal responses,decreases in affinity with TRH and methyl-histidyl-TRH correlated withincreased dissociation rates ofhormone from the receptor. Rapid dilution experiments followingsubsecond stimulation indicate thatthe TRH-R is converted rapidly from a form showing fast dissociationkinetics to a form from which thehormone dissociates slowly. Mutation of residue 105 impairs thereceptor shift between these two forms.This effect was demonstrated in a direct way by comparing[3H]methyl-histidyl-TRH dissociation ratesinCOS-7 cells transfected with either wild type or Asp105mutant TRH-Rs. Thus, residues located intransmembrane helix III positions equivalent to those of thecounterions for biogenic amines, regulatehormone-receptor interactions in the TRH receptor (and perhaps otherreceptors). Furthermore, the natureof the amino acid in these positions may also play a role, directly orindirectly, in conformational changesleading to receptor activation, and hence to signaltransduction.